Page 327 - Fundamentals of Light Microscopy and Electronic Imaging

310 IMAGE PROCESSING FOR SCIENTIFIC PUBLICATION

similarly. Remember that it will be necessary to include details on image process-
ing in the paper so that readers will know what you have done.
• Keep record sheets of imaging sessions in the research notebook. Prepare an entry
describing your acquisition parameters for each experiment. For general
microscopy, the sheet might include objective magnification, NA and type, and the
illuminator and filters used (fluorescence filter set, ND, IR- and UV-blocking fil-
ters, and others). For confocal images, notes on the pinhole size, scan rate, black-
level and gain settings, frame averaging, and step size in a z-series are important.
Some image acquisition software programs save the settings in a note associated
with the image file. It also helps to add notes about the amount of protein expres-
sion, labeling efficiency and signal strength, balance of fluorochromes, photo-
bleach rate, and other observations pertinent to each exposure. One set of notes is
usually adequate for each session involving the same preparation, but if settings
change significantly during a session, you should write them down and indicate the
image to which they pertain. It is easy to use a microscope casually without taking
notes, but the value of the images will be limited, and you are more likely to repeat
errors and waste time.
• Keep folders and directories of all computer image files so that they can be easily
reviewed and examined.

Note: Guidelines for Image Acquisition and Processing

The following guidelines are listed in the order of their occurrence from specimen
preparation to processing:

• Give priority to the quality of the specimen preparation. If the visual image does
not measure up, it is always best to prepare a better specimen in the lab rather than
to compensate for a poor image by image processing. For example, if fluores-
cence labeling is nonspecific or if there is considerable bleed-through (signal in
an image from another fluorochrome), processing the image can be difficult and
should not be encouraged. This is a particular concern when the goal is to deter-
mine if two signals are colocalized.
• Optimize image acquisition with the microscope/camera system. For fluorescence
microscopy this means proper adjustment of the microscope, illuminator, and fil-
ters for the best visible image, and full dynamic range and proper gain and offset
settings for the best acquired image. Of all the things affecting image quality,
specimen preparation and image acquisition are the most important. All too often
the offset and gain settings of the camera, and later, the histogram settings on the
computer, are used without being aware of their consequences during subsequent
image processing. There is little even an experienced computer operator can do
with an undersaturated, low-bit image.
• Display all of the specimen information in the image histogram. Before begin-
ning processing, examine the pixel values of objects and background in the
image. If you are aware of where the information is first, the danger of losing
information by adjusting the histogram will be reduced. Also check that the
image does not show evidence of clipping at white and black values, a sign that
an image has not been acquired or processed correctly. As a general rule, include